Controlling human stem cell-derived islet composition using magnetic sorting
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PROVIDER: PRJNA1168949 | ENA |
REPOSITORIES: ENA
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Project description:Stem cell-derived islets (SC-islets) hold significant promise for treating diabetes, but optimizing their functional maturity remains a challenge. CD49a, an integrin subunit involved in cell adhesion and signaling, has been identified as a potential marker associated with islet cell differentiation. In this study, we employed magnetic-activated cell sorting (MACS) to isolate CD49a-enriched and CD49a-depleted populations from SC-islets. These two distinct populations were evaluated for functional and molecular differences using single-cell RNA sequencing (scRNA-seq), in vitro glucose-stimulated insulin secretion (GSIS), and in vivo transplantation. Our scRNA-seq analysis revealed distinct transcriptional profiles between the CD49a-enriched and CD49a-depleted populations, with the CD49a-enriched population exhibiting higher expression of key β cell lineage markers, including insulin. Functional assays demonstrated that CD49a-enriched SC-islets had significantly improved GSIS, indicative of enhanced β cell functionality. Additionally, when transplanted into mice, the CD49a-enriched SC-islets exhibited superior graft performance, as evidenced by human C-peptide secretion. These findings support that CD49a enrichment through MACS represents a promising approach to enhance the functional maturity of SC-islets. Further investigations into the role of CD49a in islet cell differentiation and function may facilitate the development of more effective stem cell-based therapies for diabetes.
2025-01-28 | GSE278800 | GEO
Project description:We showed novel synthetic microRNA (miRNA) switch system, which can purify large quantities of iPSC-CMs using magnetic-activated cell sorting (MACS). We used miR-208a, which is specifically expressed in cardiomyocytes, as a specific miRNA of CMs, and CD4 as a selection marker for MACS. We synthesized a miRNA switch encoding CD4 tagged with complementary sequences against miR-208a (miR-208a CD4 switch). We transfected this switch into differentiated cells, and easily got efficiently purified CMs in a large scale with MACS. In addition, we demonstrated that purified cells were shown to be engrafted as CMs in mouse hearts.
2022-05-30 | GSE153714 | GEO
Project description:Sorting nitrifying microorganism with targeted cell sorting
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Project description:Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is often deregulated in pathological conditions, the mechanisms controlling specific RNA sorting into extracellular vescicles are still poorly understood. We identified here the RNA binding protein SYNCRIP (Synaptotagmin-binding Cytoplasmic RNA-Interacting Protein, also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP was found to bind directly a subset of miRNAs enriched in exosomes, sharing a common extra-seed sequence that we named hEXO motif. In vivo knock-down of SYNCRIP impaired microRNA sorting in exosomes and the hEXO motif was proven to play a role in the regulation of miRNA localization, as its embedment into a poorly exported miRNA enhanced its loading into exosomes. These results provide a new insight in the mechanisms of miRNA exosomal sorting process, that ca be exploited to further understand the role of these extracellular vescicles in cell-to-cell communications and the control of tissue micoenvironments.
2016-09-02 | E-MTAB-4502 | biostudies-arrayexpress
Project description:Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissociation might alter gene expressions. In this work, we cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from human pancreata. We innovated fluorescence-activated nuclei sorting (FANS) based on the positive signal of NKX2-2 antibody to enrich for nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. We observed comparable endocrine cellular composition and cell type signature gene expression between our snRNA-seq libraries and conventional scRNA-seq libraries generated with live cells from freshly isolated human islets. Our work fills a technological gap and helps to unleash archival pancreatic tissue for molecular profiling targeting the endocrine population. We expect that our protocol can be used to enrich nuclei for transcriptomics study from various populations in the pancreas and in different organs/tissues.
2024-04-01 | GSE252614 | GEO
Project description:Objective: SORCS2 is an intracellular sorting receptor genetically associated with body mass index (BMI) in humans, yet its mode of action remains unknown. Elucidating the receptor function that defines its role in metabolic health is the objective of this work. Methods: Combining in vivo metabolic studies in SORCS2-deficient mouse models with ex vivo structural and functional analyses as well as single-cell transcriptomics of murine pancreatic tissues, we studied the pathophysiological consequences of receptor dysfunction for metabolism. Results: Our studies identified an important role for SORCS2 in islet stress response essential to sustain glucose-stimulated insulin release. In detail, we show that SORCS2 is predominantly expressed in islet alpha cells. Loss of expression impairs the ability of these cells to produce osteopontin, a secreted factor that facilitates insulin release from beta cells under stress. In line with diminished osteopontin levels, beta cells in SORCS2-deficient islets show changes in gene expression related to aggravated stress, protein misfolding, as well as mitochondrial dysfunction; and they exhibit defects in insulin granule maturation and a blunted response to glucose stimulation in vivo and ex vivo. Impaired glucose tolerance in receptor mutant mice coincides with alterations in body weight and composition. Conclusion: Our data identified a novel concept in protective islet stress response involving the alpha cell receptor SORCS2, and they provide experimental support for association of SORCS2 with metabolic control in humans.
2023-05-10 | GSE231551 | GEO
Project description:The RNA binding protein SYNCRIP is a component of the exosomal machinery controlling microRNAs sorting
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Project description:Purification of Human iPSC-derived Cardiomyocytes in a Large Scale Using MicroRNA Switch and Magnetic-activated Cell Sorting
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Project description:Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic a and b cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase b cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify a, b, and d cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the sub-populations by flow cytometry and, using next generation RNA sequencing, we report on the detailed transcriptomes of fetal and adult a and b cells. We observed that human islet composition was not influenced by age, gender, or body mass index and transcripts for inflammatory gene products were noted in fetal b cells. In addition, within highly purified adult glucagon-expressing a cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet a and b cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes. RNA-sequencing of highly purified human adult and fetal islet cell subset was performed using our newly developed method. Using this data, we can study and compare the detailed transcriptome or alpha and beta cells during development.
2015-06-08 | E-GEOD-67543 | biostudies-arrayexpress
Project description:In the past decade, several transcription factors critical for pancreas development have been identified. Despite this success, many of the cell surface and extracellular factors necessary for proper islet morphogenesis and function remain uncharacterized. Previous studies have shown that transgenic over-expression of the transcription factor HNF6 specifically in the pancreatic endocrine cell lineage resulted in the disruption of islet morphogenesis, including dysfunctional endocrine cell sorting, increased islet size, and failure of islets to migrate away from the ductal epithelium. We exploited the dysmorphic islets in pdx1PBHnf6 animals as a tool to identify factors important for islet morphogenesis. Genome-wide microarray analysis was used to identify differences in the gene expression profiles of late gestation and early postnatal pancreas tissue from wild type and pdx1PBHnf6 animals. We report the identification of genes with an altered expression in HNF6 Tg animals and highlight factors with potential importance in islet morphogenesis. Keywords: disease state analysis, developmental stage analysis
2008-03-01 | GSE4926 | GEO